The large conductance Ca2+-activated K+ channel, or BKCa channel, is found in a wide variety of cell types where in general it provides feedback control over processes like neurotransmission and smooth muscle contraction that involve membrane-potential depolarization and a rise in intracellular Ca2+. Despite, however, its physiological importance, still a great deal remains to be understood about how this channel senses and responds to its primary stimuli Ca2+ and membrane voltage. In this application we propose experiments that will help remedy this situation. Three Aims are proposed. In Aim 1 we will test the prevailing hypothesis that the Ca2+-sensing mechanism of the BKCa channel is structurally similar to that of the bacterial Ca2+-activated K+ channel MthK, for which there is a crystal structure. In Aim 2, we will use electrophysiological recordings to comprehensively examine the energetics of BKCa Ca2+ sensing, and in Aim 3 we will take advantage of a recent technical advance to examine the movement of the BKCa- channel's voltage sensors exclusively when the channel is open. Results from these experiments will greatly advance our understanding of the regulation of this physiological important protein, and in so far as understanding precedes control, they may also aide in the development of pharmacological agents to be used to modulate the important physiological processes these channels regulate. PUBLIC HEALTH RELEVANCE: We propose here a series of studies that will greatly advance the understanding of the regulation of a particular ion channel protein, the large-conductance calcium-activated potassium channel, or BK channel, which plays a vital role in the regulation of neurotransmitter release in the brain and smooth-muscle contraction in the vasculature, the reproductive and digestive systems and the lungs. Because of its physiological importance, pharmacological agents that open this channel are currently being sought for the treatment of such diverse conditions as high blood pressure, stroke, asthma, impotence and incontinence. As the work proposed here is designed to uncover the natural mechanisms by which this channel is stimulated to open, it may very well point the way to the development of such beneficial pharmaceuticals.